1. Field of the Invention
The invention relates to methods and compositions for storing and distributing pesticidal or herbicidal biological organisms, in particular entomopathogenic nematodes, for use as agricultural pest control agents. More specifically, the invention concerns new methods for producing granules and for formulating nematodes into such granules for prolonged storage and convenient application.
2. Background Art
The desirability of using entomopathogenic nematodes in compositions to control pests in agricultural contexts has been clearly recognized (Gaugler et al., ENTOMOPATHOGENIC NEMATODE IN BIOLOGICAL CONTROL, CRC Press, 1990). The general classification "nematodes" includes roundworms of astounding variety ranging in size from ultramicroscopic to worms of several feet in length. In many cases, these organisms are parasitic, and their mechanisms of parasitism and their targets are as varied as their size range. While many species of nematode are themselves plant pathogens and an agricultural nuisance, a relatively benevolent group of nematodes which infect insect pests have a useful function. In particular, control of insect infestation of plants has been attempted using the "infective juvenile" (IJ) stage of these entomopathogenic nematodes. In this form, the infective juvenile enters the host insect through the alimentary canal or spiracles, emerges from its protective sheath, and penetrates into the host insect's hemocoel. There, the nematode releases symbiotic bacteria which induce septicemia that kills the host, rendering the host corpse suitable for nematode foraging and reproduction. Several generations may be spent within the insect host until food consumption and overpopulation trigger production of another IJ stage generation. The new IJs must then find fresh hosts.
In the IJ stage, the nematodes do not eat, but depend upon internal food stores; however, they do require oxygen, and, unless induced into a cryptobiotic desiccated state, require the presence of water. A major problem associated with the use of IJ nematodes as pesticides resides in the necessity to maintain the IJs in a viable state for extended time periods. The IJs cannot be kept viable simply by harvesting them from, for example, an artificial culture, and placing them in a container. The majority of the IJs so prepared will die within hours. Attempts to overcome this problem have included inducing a "cryptobiotic" state by dehydration or other means which reduce the metabolism to the point where the IJ essentially becomes inert. It is known that nematodes in the soil may exist in such a cryptobiotic state under dry climatic conditions; however, the statistics of this process are not favorable to preserving large numbers of IJs. Alternative drying means which result in a cryptobiotic state with some degree of efficiency are described, for example, in European Patent Publication No. 256,873.
In an alternative approach, the IJs are stored on various moist, high surface area materials. For example, Finney, U.S. Pat. No. 4,417,545, describes a method and package for storage and shipment of nematodes which comprises essentially a water-retaining foam sandwich. While the container is stated to be "suitable for storing nematodes", only nematode eggs were tested. Notably, only one particular foam was found to be successful in maintaining the viability of the eggs, even when storage was at temperatures as low as -5.degree. C.
Yukawa, U.S. Pat. No. 4,765,275 describes a packaging system for nematode storage and transport, wherein nematodes are homogeneously mixed with materials described as adsorbents (e.g., activated charcoal) and stored as a suspension under conditions which prevent microbial growth.
Nelsen et al., U.S. Pat. No. 4,615,883, describes a formulation in which the IJs are encapsulated in an alginate gel obtained by adding calcium ions to a sodium alginate suspension of the nematodes. In these preparations, capsules of 0.5-5 mm in diameter are formed and are said to improve the storageability of the IJs. The use of alginate gels and other encapsulating materials to encapsulate living tissue or cells (though not specifically IJs) has an extensive literature. See, for example, U.S. Pat. Nos. 4,409,331; 4,407,957; 4,391,909; 4,352,883; 4,663,286; 4,778,749; 4,798,786; 4,803,168; 4,806,355; 4,647,536; and 4,814,274. All of these documents describe methods to encapsulate living cells or tissues in various polymeric capsules. The purpose of the capsules in each case is to preserve the viability of the living tissue and, also, to facilitate the use of such tissue in its intended application.
U.S. patent application Ser. No. 07/313,594, filed Feb. 21, 1989, abandoned, assigned to the same assignee and incorporated herein by reference, describes a packaging method for IJs in a reversibly cross-linked matrix which is embedded in a supporting screen. The screen containing the sheet matrix is simply placed in the location of end use, and application of water and an active decross-linking agent liberates the embedded nematodes from the film.
U.S. Pat. No. 5,170,744 (Dec. 15, 1992), commonly assigned with the present application, discloses a more convenient and effective manner of preserving viability of IJ nematodes by immobilizing them in a pseudoplastic gel which can be dispensed in the field by applying a suitable shear force. While embedded in the matrix, the nematodes are immobilized, resulting in reduced metabolism and improved viability. However, upon liquefaction of the supporting medium, the nematodes can be readily dispensed in the desired location.
Bedding, U.S. Pat. No. 5,042,427 discloses a range of product types obtained by mixing IJs with an absorbent material (clay) to remove excess surface moisture and to produce partial desiccation. Homogeneous blends as well as non-homogeneous blends of nematodes and absorbents are described, such as a sandwich consisting of a layer of nematodes between two layers of clay. Granules are not disclosed. These products lack the convenience of either water dispersible or directly applied granules. The clay layers are said to be about two centimeters thick, which would appear to serve as a powerful barrier to oxygen diffusion, thus raising questions about adequate oxygenation of the nematodes using this formulation.
Other work discloses methods of producing starch granules containing various biocontrol agents, such as bacteria; nematodes were not specified (U.S. Pat. No. 4,859,377).
Capinera et al., J. Agric. Entomol. (1987) 4:337) described a pellet nematode formulation which pellets contained alfalfa meal, wheat flour and other components.
Connick et al. (1991) Biological Control 1:281-287, disclosed entrapment of mycoherbicidal fungi in a pasta-like wheat flour dough. The dough, used for weed control, was air-dried and ground into granules which were sized by sieving.
Connick et al., U.S. patent application Ser. No. 07/560,792 (filed Jul. 9, 1990, licensed to the U.S. Agricultural Research Service), describes an extruded or formed granular product in which nematodes are uniformly distributed throughout a wheat gluten matrix. The formulation also included a filler and humectant to enhance nematode survival. Granules are then dried to low moisture to prevent nematode migration and to reduce risk of microbial contamination. Dried granules are very difficult to dissolve; only partial dissolution was achieved, even after submerging of granules in water for 48 hours. Reported nematode survival rates were relatively low. Further, it appears that this product is limited to direct soil application and cannot be used in spray applications.
In many of the above disclosed formulations, the nematodes or other organisms are homogeneously distributed throughout the composition. This is a source of problems, as discussed below, which the present invention has overcome.
A serious problem in the art not yet successfully overcome is that of nematode migration from the formulated preparations currently on the market. For example, nematodes migrate freely from Connick's `pasta` granules (Connick et al., supra) unless they are dried sufficiently to inactivate the nematodes. Nematodes that migrate to a surface do not typically survive for long. Once at the surface, nematodes will often assume an adaptive behavior known as "nictation", which is the standing upon one end and actively waving their bodies in the air. Nictation dramatically increases metabolic rate, as measured by oxygen consumption, thereby decreasing the length of time that the initial fixed amount of energy reserves will last before depletion. Furthermore, drying of nematodes becomes a significant problem once they are directly exposed to air. With many of the commercially valuable nematodes such as Steinernema carpocapsae, air drying leads to death. Further still, nematodes will migrate from the surface of the product and onto, and potentially out of, the packaging in which they were originally contained. Nematodes that leave the safety of their formulated product rapidly succumb to fatal dehydration. The present invention is directed in part to such problems in the art.
Disclosure of the Invention
The invention provides compositions and methods for their production, which compositions are granules encasing biological organisms useful as herbicides or pesticides, in particular beneficial infective juvenile nematodes. The composition and packaging means therefor are useful for storage, shipping, and dispensing. The methods and formulations provided offer convenience and effectiveness and are suitable for large-scale production, long term storage, and distribution of nematode preparations.
The present invention is directed to a storage composition of pesticidal or herbicidal viable biological organisms comprising a granule formed from a powder or mixture of powders having encased therein a soft core containing a quantity of viable biological organisms.
The organisms are preferably maintained in a partially desiccated state, thereby reducing metabolic activity and extending survival in storage. The degree of desiccation can be controlled by appropriate selection of the dry powder composition and the processing mathods.
In the above composition, the powder is preferably selected from the group consisting of a silica, a diatomaceous earth, hydroxyethyl cellulose, a clay, a pregelled clay, a lignosulfonate, a modified starch, a pregelatinized starch, a superabsorbent and a combination of one or more thereof.
The above composition may further contain one or more of an antibacterial agent, an antifungal agent, a humectant, a stabilizing agent, a viscosifier, a wetting agent or other surfactant, and an ultraviolet light-absorbing agent, all of which agents are not toxic to the biological organisms.
In a preferred embodiment, the biological organisms of the composition are beneficial nematodes, most preferably entomopathogenic nematodes. Preferably the nematodes are infective juvenile nematodes. Preferred entomopathogenic nematodes are of the family Steinernematidae or Heterorhabditidae. In another embodiment, the nematode is a molluskicidal nematode of the family Phasmarhabditidae.
Where the organisms are nematodes, the suspension contains nematodes at a concentration of preferably about 10.sup.5 to about 2.times.10.sup.6 per gram of suspension, more preferably about 8.times.10.sup.5 to 10.sup.6 per gram.
The present invention is also directed to a method for preparing a storage composition of pesticidal or herbicidal viable biological organisms, comprising contacting individual drops or droplets of an aqueous suspension containing the organisms with a powder such that the powder completely surrounds each of the drops, wherein moisture within the drops is drawn into the powder causing the powder to form a solidified casing surrounding the drops containing the organism.
The organisms are preferably beneficial nematodes, more preferably infective juvenile entomopathogenic nematodes, preferably of the family Steinernematidae or Heterorhabditidae.
In the above method, the suspension preferably contains nematodes at a concentration of about 10.sup.5 to about 2.times.10.sup.6 per gram of suspension, more preferably at a concentration of about 8.times.10.sup.5 to 10.sup.6 per gram.
In the above methods, the contacting may be performed by manually applying the drops to the powder. The powder is preferably in motion, either rotational or vibrational at the time of the contacting. In another embodiment, the contacting is performed by spraying droplets of the suspension into a falling curtain of powder. The curtain may fall onto a rotating or vibrating surface on which surface consolidation of granules takes place. In another embodiment, the contacting is performed by applying droplets to a powder suspended in an air-suspended fluidized bed or counter-current fluidized bed apparatus.
In the above methods, the powder is preferably selected from the group consisting of a silica, a clay, a pregelled clay, a diatomaceous earth, a hydroxyethyl cellulose, a lignosulfonate, a modified starch, a pregelatinized starch, a superabsorbent and a combination of one or more thereof.
The present invention is further directed to a method of controlling pests, preferably insects, comprising applying a composition as described above to a plant or field to control the pests. The applying may be performed prior to signs of pest infestation to prevent the infestation or after such infestation. In this method, the nematodes are preferably of the family Steinernematidae, Heterorhabditidae or Phasmarhabditidae.
In the method of controlling pests, the composition may be applied as granules to soil or dissolved in water and applied as a liquid, preferably as a spray to soil or to plants.